Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02859627 2014-06-17
DESCRIPTION
[Title of the Invention]
Novel Anti-Human CTGF Antibody
[Technical Field]
[0001]
The present invention relates to a novel anti-human CTGF antibody.
Specifically,
the novel anti-human CTGF antibody of the present invention is an anti-human
CTGF
antibody having excellent binding activity and/or neutralizing activity, as
compared with
conventional anti-human CTGF antibodies.
[Background Art]
[0002]
CTGF (connective tissue growth factor) is a secreted protein rich in cysteine
residues with a molecular weight of about 36 to 38 kDa, belonging to a CCN
family (Non-
Patent Document 1), and has been conventionally known to be induced by TGF-I3
that can
be considered to be the most important growth factor in fibrosis (Non-Patent
Document 2).
Therefore, it is suggested that TGF-f3 induces CTGF and the induced CTGF
promotes the
fibrosis of organs or tissues, and it is believed that CTGF plays an important
role in
fibrosis, cell proliferation, metabolism of the extracellular matrix,
angiogenesis,
arteriosclerosis, and the like (Non-Patent Document 3).
[0003]
It has become known that there are many domains present in CTGF, which
interact with other factors. Among them, it is known that CTGF is coupled
directly with
TGF-P or BMP4 via von Willebrand C domain, and causes the promotion of TGF-I3
signaling or the inhibition of BMP signaling (Non-Patent Document 4).
[0004]
It has become known that expression of CTGF is increased in various renal
diseases (for example, chronic kidney disease, diabetic nephropathy,
glomerulosclerosis,
IgA nephropathy, focal segmental glomerulosclerosis, ANCA-related nephritis,
acute
progressive glomerulonephritis, chronic transplant nephropathy, nephrotic
syndrome, lupus
nephritis and membranoproliferative glomerulonephritis) (Non-Patent Document
5), and it
has been reported that CTGF is deeply involved in fibrosis (Non-Patent
Document 6).
[0005]
In addition, it has been reported that CTGF is involved in various types of
fibrosis
(scleroderma, interstitial lung disease, pulmonary fibrosis diseases such as
idiopathic
pulmonary fibrosis, fibrosis caused by chronic hepatitis B or C, radiation-
induced fibrosis,
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fibrosis caused by wound healing, and cardiac hypertrophy and fibrosis),
vascular
proliferative diseases, diabetic retinopathy, cancer, and the like, and thus,
it can be thought
that CTGF could be a new therapeutic target (Non-Patent Documents 7 and 8).
[0006]
Therefore, if a monoclonal antibody which specifically binds to CTGF and has
an
activity inhibiting various actions of CTGF can be developed, the monoclonal
antibody is
expected to be useful for diagnosis, prevention or treatment of various
diseases in which
CTGF is involved in pathogenesis.
[0007]
As an antibody showing an inhibitory function against human CTGF, which have
been hitherto studied, human monoclonal antibodies M84 and M320 (Patent
Document 1),
CLN1 (Patent Document 2), a mouse monoclonal antibody CTGF-m2-1 (Patent
Document
3), and the like have been reported. Among them, CLNI has been investigated in
most
detail, and its effect has been identified in an interstitial pulmonary
fibrosis model or a
renal interstitial fibrosis model by unilateral ureteral ligation. CLNI is
studied in clinical
trial (Phase II) as FG-3019.
[0008]
However, it cannot be said that conventional antibodies have sufficient
binding
activity for CTGF, and have sufficiently strong neutralizing activity for CTGF
from a
[0009]
In general, examples of the major factors defining the effective doses of the
antibody pharmaceuticals include the binding activity or neutralizing activity
which the
antibody has for an antigen, and the amount of an antigen present in the body.
However,
[0010]
For these reasons, it is essential to acquire an anti-human CTGF antibody
having
[Related Art]
[0011]
[Patent Document 1] JP-A-2000-232884
[Patent Document 2] W02004/108764
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[Patent Document 3] W02007/066823
[Non-Patent Document]
[0012]
[Non-Patent Document 1] D.M. Bradham et al., J. Cell Biol. 114:1285-1294
(1991)
[Non-Patent Document 2] A. Igarashi et al., Mol. Biol. Cell 4:637-645 (1993)
[Non-Patent Document 3] Blom IE et al., Matrix Biol. 21(6):473-82 (2002)
[Non-Patent Document 4] Abreu, et al., Nat. Cell. Biol. 4, 599-604 (2002)
[Non-Patent Document 5] Ito Y et at., Kidney Int. 53(4) 853-61 (1998)
[Non-Patent Document 6] Phanish MK et al., Nephron Exp Nephrol. 114(3) e83-
92 (2010)
[Non-Patent Document 7] Shi-Wen X et at., Cytokine Growth Factor Rev.
19(2):133-44 (2008)
[Non-Patent Document 8] Jun JI et al., Nat Rev Drug Discov. 10(12):945-63
[Disclosure of Invention]
[Problem to Be Solved by the Invention]
[0013]
An object of the present invention is to provide anti-human CTGF antibodies
having excellent binding activity and/or neutralizing activity, as compared
with
conventional anti-human CTGF antibodies.
[Means for Solving the Problems]
[0014]
The present invention includes the following invention as medically or
industrially useful substances and methods.
[1] An anti-human CTGF antibody, comprising:
a heavy-chain variable region consisting of the amino acid sequence shown by
a light-chain variable region consisting of the amino acid sequence shown by
SEQ
ID NO: 4.
[2] The anti-human CTGF antibody according to [1], wherein a heavy-chain
constant region of the antibody is a human Igyl constant region.
[3] The anti-human CTGF antibody according to [1], wherein a light-chain
constant region of the antibody is a human Igic constant region.
[4] The anti-human CTGF antibody according to [1], wherein a heavy-chain
constant region of the antibody is a human Igyl constant region, and a light-
chain constant
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region of the antibody is a human Igx constant region.
[5] The anti-human CTGF antibody according to [1], comprising:
a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 12;
and
a light chain consisting of the amino acid sequence shown by SEQ ID NO: 8.
[6] A polynucleotide comprising a sequence that encodes the heavy-chain
variable region of the antibody according to any one of [1] to [5].
[7] A polynucleotide comprising a sequence that encodes the light-chain
variable region of the antibody according to any one of [1] to [5].
[8] An expression vector comprising the polynucleotide according to [6] and/or
[7].
[9] A host cell transformed with the expression vector according to [8].
[10] The host cell according to [9], which is selected from the group
consisting
of the following (a) and (b):
(a) a host cell transformed with an expression vector comprising a
polynucleotide
comprising a sequence that encodes the heavy-chain variable region of the
antibody
according to any one of [1] to [5] and a polynucleotide comprising a sequence
that encodes
the light-chain variable region of the antibody; and
(b) a host cell transformed with an expression vector comprising a
polynucleotide
[11] A method for producing the anti-human CTGF antibody according to any
one of [1] to [5], the method comprising expressing the anti-human CTGF
antibody by
[12] An therapeutic agent for a disease in which human CTGF is involved in
pathogenesis, comprising the antibody according to any one of [1] to [5].
[13] The therapeutic agent according to [12], wherein the disease is kidney
disease.
30 [14] The therapeucitc agent according to [13], wherein the kidney
disease is
chronic kidney disease or diabetic nephropathy.
[15] A method for preventing or treating a disease in which human CTGF is
involved in pathogenesis, comprising administering the antibody according to
any one of
[1] to [5].
35 [16] The method according to [15], wherein the disease is kidney
disease.
[17] The method according to [16], wherein the kidney disease is chronic
kidney
disease or diabetic nephropathy.
[18] The antibody according to any one of [1] to [5], for use in preventing or
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treating a disease in which human CTGF is involved in pathogenesis.
[19] The antibody according to [18], wherein the disease is kidney disease.
[20] The antibody according to [19], wherein the kidney disease is chronic
kidney disease or diabetic nephropathy.
[Effects of the Invention]
[0015]
According to the present invention, anti-human CTGF antibodies having
excellent
binding activity and/or neutralizing activity, as compared with conventional
anti-human
CTGF antibodies, are provided. The anti-human CTGF antibody of the present
invention
has a potent antifibrotic action by inhibiting the function of human CTGF, and
is useful for
prevention or treatment of various diseases, in which human CTGF is involved
in
pathogenesis. Further, the anti-human CTGF antibody of the present invention
provides
significant improvements in clinical applications such as reduction of dosage,
extension of
administration interval, improvement of the mode of administration (for
example, a
subcutaneous injection), and the like, and thus, greatly contributes to
improvement in
treatment effectiveness and patient compliance.
[Embodiments for Carrying Out the Invention]
[0016]
Hereinafter, the present invention will be described in detail.
The present inventors have made extensive studies on preparation of anti-human
CTGF antibodies, and as a result, they have succeeded in producing anti-human
CTGF
antibodies having improved binding activity and excellent neutralizing
activity as
compared with conventional anti-human CTGF antibodies.
[0017]
The basic structure of an antibody molecule is shared amongst all antibody
classes, and is configured with a heavy chain having a molecular weight of
50000 to 70000
and a light chain having a molecular weight of 20000 to 30000. The heavy chain
usually
consists of a polypeptide chain comprising about 440 amino acids. Heavy chains
have
structures characteristic of different classes, and are called the 7, j.i, a,
8 and c chains
corresponding to IgG, IgM, IgA, IgD and IgE. Furthermore, IgG occurs as IgG 1,
IgG2,
IgG3 and IgG4, and the corresponding chains are called 71, y2, 73 and y4,
respectively. A
light chain usually consists of a polypeptide chain comprising about 220 amino
acids, two
types of which, type L and type K, are known, and are called the and lc
chains,
respectively. Regarding the peptide configuration of the basic structure of an
antibody
molecule, two homologous heavy chains and two homologous light chains are
bound via
disulfide bonds (S-S bonds) and non-covalent bonds, and the molecular weight
is 150000
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to 190000. The two kinds of light chains are capable of pairing with any heavy
chain.
Each antibody molecule always consists of two identical light chains and two
identical
heavy chains.
[0018]
There are four intrachain S-S bonds in a heavy chain (five bonds for and
chains) and two in a light chain; one loop is formed per 100 to 110 amino acid
residues,
and this steric structure is alike among the loops, and is called a structural
unit or domain.
For both heavy chains and light chains, the amino acid sequence of the domain
located at
the N terminus thereof is not constant, even in a reference standard from the
same class
(subclass) of the same animal species, and this domain is called the variable
region. Each
of the domains is called a heavy-chain variable region (VH) and a light-chain
variable
region (VI), respectively. The amino acid sequence on the C-terminal side
therefrom is
nearly constant in each class or subclass, and is called a constant region
(each of the
domains is called CHI, CH2, CH3 and CL, respectively).
[0019]
The antigenic determinant site of an antibody is configured with Vii and VL,
and
the binding specificity depends on the amino acid sequence of this site. On
the other
hand, biological activities such as binding to complements or various cells
reflect the
differences in the constant region structure among the various classes of Ig.
The
variability in the variable regions of the light chain and heavy chains is
mostly limited to
three small hypervariable regions existing in both chains, and these regions
are called
complementarity determining regions (CDRs; CDR1, CDR2 and CDR3 starting from
the
N-terminal side). The remaining portion of the variable region is called a
framework
region (FR) and is relatively constant.
[0020]
The anti-human CTGF antibody of the present invention that the present
inventors
have succeeded in preparing is an anti-human CTGF antibody having the
following
characteristics.
An anti-human CTGF antibody comprising a heavy-chain variable region
[0021]
Specifically, the present inventors constructed antibodies using a human
monoclonal antibody development technology, "VelocImmune " mouse [VelocImmune
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heavy chain and light chain variable regions are replaced with the
corresponding human
variable regions are immunized with the antigen of interest (for example,
human CTGF),
and lymphatic cells are recovered from the mice that express antibodies. The
lymphatic
cells are fused with mouse myeloma cells to prepare hybridomas. The hybridoma
cells
are screened to identify hybridoma cells that produce those antibodies that
specifically bind
to the antigen of interest. The antibodies that are produced herein are
antibodies having
the variable regions of human antibodies and the constant regions of mouse
antibodies
(also referred to as chimeric antibodies). Then, if the antibody that binds
specifically to
the antigen of interest are identified, DNAs that encode the variable regions
of the heavy
chain and light chain of the antibody are isolated from the hybridoma cells
and linked to
DNAs encoding the constant regions of the heavy chain and light chain of a
desired class
of human antibody, respectively. The resulting gene encoding the heavy chain
and light
chain of the antibody is expressed in cells (e.g., CHO cells) to produce an
antibody
molecule. The heavy chain and light chain of the antibody produced by the
above method
are the heavy chain and light chain of a "fully human" antibody derived from a
human
immunoglobulin gene.
[0022]
The anti-human CTGF antibody of the present invention can be easily prepared,
based on the information on the sequence of the heavy-chain variable region
and the light-
chain variable region as disclosed in the present specification, using methods
known in the
art, by a person skilled in the art. Preferably, the anti-human CTGF antibody
of the
present invention can be prepared as a fully human antibody by linking the
heavy-chain
variable region and the light-chain variable region to a heavy-chain constant
region and a
light-chain constant region of a human antibody, respectively. Specifically, a
heavy-chain
variable region gene fragment having a base sequence encoding the heavy-chain
variable
region amino acid (SEQ ID NO: 10) of the antibody of the present invention and
a light-
chain variable region gene fragment having a base sequence encoding the light-
chain
variable region amino acid (SEQ ID NO: 4) of the antibody of the present
invention are
prepared. Further, these variable region genes are linked to a suitable class
of constant
region genes of a human antibody to prepare a fully human antibody gene.
Subsequently,
this antibody gene is linked to a suitable expression vector and introduced
into cultured
cells. Finally, the cultured cells are cultured and a monoclonal antibody can
be obtained
from the culture supernatant.
[0023]
The gene fragments that encode the heavy-chain variable region and light-chain
variable region amino acids of the antibody of the present invention can be
synthesized
using a gene synthesis method known in the art, on the basis of, for example,
base
sequences designed based on the amino acid sequences of the heavy chain and
light chain
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variable regions. As such gene synthesis method, various methods known to
those skilled
in the art, such as the antibody gene synthesis method described in
W090/07861, can be
used.
[0024]
Then, the above-described variable region gene fragments are linked to the
constant region genes of the human antibody to prepare a fully human antibody
gene.
Although any subclass of the constant region (for example, the constant region
of a heavy
chain such as the y1, y2, y3 or y4 chain, and the constant region of a light
chain such as the
k or lc chain) can be chosen as the constant region of the human antibody
used, human Igy 1
as the heavy-chain constant region, and human Igic as the light-chain constant
region, can
preferably be used.
[0025]
Subsequent to the preparation of this fully human antibody gene, introduction
of
the antibody gene into an expression vector, introduction of the expression
vector into
cultured cells, cultivation of the cultured cells, purification of the
antibody and the like can
be performed using various methods known in the art.
[0026]
An expression vector that is linked to the antibody gene thus obtained
includes GS
vector pEE6.4 or pEE12.4 (Lonza Biologics), but are not specifically limited,
so long as
they can express such antibody gene. Also, an expression vector already having
a human
Ig constant region gene such as AG-y1 or AG-ic (for example, see W094/20632)
may be
used.
[0027]
The above-described expression vector is introduced into cultured cells by,
for
[0028]
As cultured cells into which the expression vector is introduced, cultured
cells
such as CHO-K 1 SV cells, CHO-DG44 cells and 293 cells can be used, and these
cells may
be cultured by a conventional method.
[0029]
After the above-described culture, the antibody accumulated in the culture
supernatant can be purified by various column chromatography, for example,
various
chromatographic processes using a Protein A or Protein G column.
[0030]
The anti-human CTGF antibody of the present invention is an antibody which
binds to human CTGF. Examples of a method for measuring the binding activity
of the
obtained anti-human CTGF antibody for human CTGF include an ELISA method and a
surface plasmon resonance (SPR) analysis method. For example, when ELISA is
used,
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human CTGF (SEQ ID NO: 14) is immobilized onto an ELISA plate, and the anti-
human
CTGF antibody is added thereto and allowed to react therewith. Then, the
resultant is
allowed to react with a secondary antibody such as an anti-IgG antibody
labeled with an
enzyme such as horseradish peroxidase (HRP), and washed. Then, the absorbance
is
measured by adding a chromogenic substrate (for example, a TMB chromogenic
reagent in
the case of HRP labeling). Further, the binding activity for the human CTGF
can be
measured in more detail using SPR analysis. When SPR analysis is carried out,
for
example, a Biacore system can be used to measure the association rate constant
(ka) and
the dissociation rate constant (kd) between the anti-human CTGF antibody and
the human
CTGF, and thus, a dissociation constant (KD) can be calculated from the ratio
of the two
constants. The anti-human CTGF antibody of the present invention also includes
an
antibody which also binds to CTGF derived from other animals (for example,
mouse
CTGF), and the binding activity thereof for protein may also be measured.
[0031]
Furthermore, the anti-human CTGF antibody of the present invention has
neutralizing activity for human CTGF. As used in the present specification,
the
"neutralizing activity" of the antibody means an activity to inhibit any
biological activity
resulting from CTGF by the binding to CTGF, and can be evaluated on one or
more
biological activities of CTGF as an index. Examples of such neutralizing
activity include
an inhibitory action against collagen synthesis in fibroblasts derived from
the kidney
(inhibition of fibrosis), and the neutralizing activity can be evaluated using
a method as
described in Examples below.
[0032]
In order to evaluate the effects of the anti-human CTGF antibody of the
present
invention in more detail, a test on the efficacy of the antibody in vivo can
also be used.
For example, by evaluating the function of the kidney using a mouse model with
chronic
kidney disease or a rat model with nephritis as described in Examples below,
the efficacy
of the antibody in vivo can be evaluated.
[0033]
In addition, methods for evaluating various types of stability (for example,
thermal stability, long-term storage stability and high-concentration
stability) of the anti-
human CTGF antibody of the present invention include differential scanning
calorimetry
and a method of measuring the formation of aggregates during the storage.
[0034]
Preferably, the anti-human CTGF antibody of the present invention can be
easily
acquired by synthesizing DNA comprising a base sequence encoding the heavy-
chain
variable region amino acid sequence shown by SEQ ID NO: 10 and DNA comprising
a
base sequence encoding the light-chain variable region amino acid sequence
shown by
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SEQ ID NO: 4, and linking these DNAs to a suitable class of human antibody
constant
region genes, preferably a human Igyl constant region gene for the heavy chain
and a
human Igic constant region gene for the light chain, so as to construct a
fully human
antibody gene by using a method known in the art, and introducing the fully
human
antibody gene into an expression vector, introducing the expression vector
into a cultured
cell, culturing the cultured cell, and purifying an antibody from the obtained
culture by
using various methods known in the art. Preferably, the DNA comprising the
base
sequence encoding the heavy-chain variable region amino acid sequence shown by
SEQ ID
NO: 10 comprises the base sequence shown by SEQ ID NO: 9. Preferably, the DNA
comprising the base sequence encoding the light-chain variable region amino
acid
sequence shown by SEQ ID NO: 4 comprises the base sequence shown by SEQ ID NO:
3.
[0035]
A preferred heavy chain of anti-human CTGF antibody of the present invention,
comprising the heavy-chain variable region shown by SEQ ID NO: 10 and a human
Igy 1
constant region, is a heavy chain consisting of the amino acid sequence shown
by SEQ ID
NO: 12. A preferred light chain of anti-human CTGF antibody of the present
invention,
comprising the light-chain variable region shown by SEQ ID NO: 4 and a human
Igic
constant region, is a light chain consisting of the amino acid sequence shown
by SEQ ID
NO: 8. Preferably, DNA comprising a base sequence encoding an anti-human CTGF
antibody heavy chain consisting of the amino acid sequence shown by SEQ ID NO:
12
comprises the base sequence shown by SEQ ID NO: 11. Preferably, DNA comprising
a
base sequence encoding an anti-human CTGF antibody light chain consisting of
the amino
acid sequence shown by SEQ ID NO: 8 comprises the base sequence shown by SEQ
ID
NO: 7. An anti-human CTGF antibody of the present invention, which comprises a
heavy
chain consisting of the amino acid sequence shown by SEQ ID NO: 12 and a light
chain
consisting of the amino acid sequence shown by SEQ ID NO: 8, includes a fully
human
37-45-MH1 as described in Examples below.
[0036]
The present invention also comprises an anti-human CTGF antibody that
comprises a heavy-chain variable region comprising CDR1 consisting of amino
acid
sequence at position from 31 to 35 of SEQ ID NO: 10, CDR2 consisting of amino
acid
sequence at position from 50 to 66 of SEQ ID NO: 10, and CDR3 consisting of
amino acid
sequence at position from 99 to 108 of SEQ ID NO: 10, and a light-chain
variable region
comprising CDR1 consisting of amino acid sequence at position from 24 to 35 of
SEQ ID
NO: 4, CDR2 consisting of amino acid sequence at position from 51 to 57 of SEQ
ID NO:
4, and CDR3 consisting of amino acid sequence at position from 90 to 98 of SEQ
ID NO:
4. The anti-human CTGF antibody can be also prepared by those skilled in the
art
according to procedures such as ones described above.
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[0037]
The present invention also comprises anti-human CTGF antibody fragments such
as a single-chain variable region fragment (scFv), Fab, Fab' and F(ab')2,
which comprise
the heavy-chain variable region and light-chain variable region of the
antibody of the
present invention and maintain the activity of the antibody. Any person
skilled in the art
can construct a fusion antibody of the anti-human CTGF antibody or antibody
fragment
and another peptide or protein and can also construct a modified antibody
having a
modifying agent bound thereto, on the basis of the present invention. The
other peptide
or protein used for the fusion is not specifically limited, so long as it does
not reduce the
binding activity of the antibody; examples thereof include human serum
albumin, various
tag peptides, artificial helix motif peptide, maltose-binding proteins,
glutathione S
transferase, various toxins, other peptides or proteins capable of promoting
multimerization,
and the like. The modifying agent used for the modification is not
specifically limited, so
long as it does not reduce the binding activity of the antibody; examples
thereof include
polyethylene glycol, sugar chains, phospholipids, liposomes, low-molecular
compounds
and the like.
[0038]
The anti-human CTGF antibody of the present invention thus obtained may be
further purified as necessary, and may be then formulated according to an
ordinary method,
and thus it can be used for prevention or treatment of diseases in which CTGF
is involved
in pathogenesis, such as renal diseases such as chronic kidney disease and
diabetic
nephropathy, vascular proliferative diseases, cardiomyopathy, hepatic
fibroplasia disease,
pulmonary fibrosis, skin fibrosis disease, diabetic retinopathy and cancer.
[0039]
The anti-human CTGF antibody of the present invention can be preferably used
as
a therapeutic agent for kidney diseases, and more preferably a therapeutic
agent for chronic
kidney disease or diabetic nephropathy. Examples of the formulations for these
therapeutic agents or the like include parenteral agents such as an injection
agent and an
infusion agent, and administration thereof using intravenous administration,
subcutaneous
administration, or the like is preferred. In addition, for the formulation,
within a
pharmaceutically acceptable range, a carrier or an additive can be used
according to these
formulations.
[0040]
The amount of the anti-human CTGF antibody of the present invention added in
the above-described formulation varies depending on the patient's symptom
severity or age,
the dosage form of the formulation used or the binding titer of the antibody
and the like;
for example, about 0.001 mg/kg to 100 mg/kg of the antibody may be used.
[0041]
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The present invention also provides a polynucleotide comprising a sequence
encoding an anti-human CTGF antibody of the present invention, and an
expression vector
comprising the same. The present invention also provides a polynucleotide
comprising a
sequence encoding the heavy-chain variable region of the anti-human CTGF
antibody of
the present invention, and a polynucleotide comprising a sequence encoding the
light-chain
variable region of the anti-human CTGF antibody of the present invention, and
expression
vector comprising either or both of them. The expression vector of the present
invention
is not specifically limited, so long as it can express a gene that encodes the
antibody of the
present invention or its heavy-chain variable region and/or light-chain
variable region in
various host cells of prokaryotic cells and/or eukaryotic cells, and produce
these
polypeptides. Examples thereof include plasmid vectors, viral vectors (for
example,
adenovirus, retrovirus) and the like. Preferably, the expression vector of the
present
invention comprises a polynucleotide comprising either a sequence encoding the
heavy
chain or light chain of the above-described antibody of the present invention,
or both a
polynucleotide comprising a sequence encoding the heavy chain of the antibody
of the
present invention and a polynucleotide comprising a sequence encoding the
light chain of
the antibody of the present invention.
[0042]
The expression vector of the present invention can comprise a promoter
operably
linked to a gene that encodes the anti-human CTGF antibody of the present
invention or its
heavy-chain variable region and/or light-chain variable region. A promoter for
expressing
a gene encoding the antibody of the present invention or its heavy-chain
variable region
and/or light-chain variable region in a bacterium includes, for example, Trp
promoter, lac
promoter, recA promoter, kPL promoter, 1pp promoter, tac promoter and the
like, when the
host is a bacterium of the genus Escherichia. A promoter for expression in
yeast includes,
for example, PHO5 promoter, PGK promoter, GAP promoter and ADH promoter, and
some
examples of a promoter for expression in the genus Bacillus include SLO1
promoter, SPO2
promoter, penP promoter and the like. When the host is a eukaryotic cell such
as a
mammalian cell, a promoter includes SV40-derived promoter, retrovirus
promoter, heat
shock promoter and the like.
[0043]
When a bacterium, particularly Escherichia coli, is used as the host cell, the
expression vector of the present invention can further comprise an initiation
codon, a stop
codon, a terminator region and a replicable unit. When yeast, an animal cell
or insect cell
is used as the host, the expression vector of the present invention can
comprise an initiation
codon and a stop codon. In this case, it may comprise an enhancer sequence,
noncoding
regions on the 5' side and 3' side of a gene that encodes the antibody of the
present
invention or its heavy-chain variable region or light-chain variable region, a
secretion
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signal sequence, a splicing junction, a polyadenylation region, a replicable
unit or the like.
Also, it may comprise a selection marker that is in common use (for example,
tetracycline-
resistant gene, ampicillin-resistant gene, kanamycin-resistant gene, neomycin-
resistant
gene, dihydrofolic acid reductase gene) according to the intended use.
[0044]
The present invention also provides a transformant introduced with a gene
encoding the antibody of the present invention or its heavy-chain variable
region and/or
light-chain variable region. Such a transformant can be prepared by, for
example,
transforming a host cell with the expression vector of the present invention.
A host cell
.. that is used to prepare the transformant is not specifically limited, so
long as it is suitable
for the aforementioned expression vector and is transformable; examples
thereof include
various cells such as natural cells or artificially established cells commonly
being used in
the technical field of the present invention (for example, bacteria (bacteria
of the genus
Escherichia, bacteria of the genus Bacillus), yeasts (the genus Saccharomyces,
the genus
.. Pichia and the like), animal cells or insect cells (for example, Sf9) and
the like. The
transformation can be performed by any known method per se.
[0045]
Preferably, the transformant of the present invention is a host cell
transformed
with an expression vector comprising a polynucleotide comprising a sequence
encoding
.. the heavy-chain variable region of the antibody of the present invention
and a
polynucleotide comprising a sequence encoding the light-chain variable region
of the
antibody of the present invention, or a host cell transformed with an
expression vector
comprising a polynucleotide comprising a sequence encoding the heavy-chain
variable
region of the antibody of the present invention and an expression vector
comprising a
.. polynucleotide comprising a sequence encoding the light-chain variable
region of the
antibody of the present invention. More preferably, the transformant of the
present
invention is a host cell transformed with an expression vector comprising a
polynucleotide
comprising a sequence encoding the heavy chain of the antibody of the present
invention
as described above and a polynucleotide comprising a sequence encoding the
light chain of
.. the antibody of the present invention, or a host cell transformed with an
expression vector
comprising a polynucleotide comprising a sequence encoding the heavy chain of
the
antibody of the present invention as described above and an expression vector
comprising
a polynucleotide comprising a sequence encoding the light chain of the
antibody of the
present invention.
[0046]
The present invention further provides a method for producing the anti-human
CTGF antibody of the present invention, comprising expressing a gene encoding
the
antibody of the present invention or the heavy-chain variable region and/or
the light-chain
13
CA 02859627 2014-06-17
variable region thereof in a host cell, that is, using such a transformant.
Preferably, the
host cell used in the method is a host cell transformed with the expression
vector of the
present invention as described above, and the expression vector may comprise a
polynucleotide comprising a sequence encoding the heavy-chain variable region
of the
antibody of the present invention and a polynucleotide comprising a sequence
encoding the
light-chain variable region of the antibody of the present invention,
separately or
simultaneously.
[0047]
When producing the anti-human CTGF antibody of the present invention, the
transformant may be cultured in a nutrient medium. The nutrient medium
preferably
contains a carbon source and an inorganic nitrogen source or organic nitrogen
source,
which are required for the growth of the transformant. Examples of the carbon
source
include glucose, dextran, soluble starch, sucrose and the like; examples of
the inorganic
nitrogen source or organic nitrogen source include ammonium salts, nitrates,
amino acids,
corn steep liquor, peptone, casein, meat extract, soybean cake, potato extract
and the like.
If desired, other nutrients (for example, inorganic salts (for example,
calcium chloride,
sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (for
example,
tetracycline, neomycin, ampicillin, kanamycin and the like) and the like) may
be contained.
[0048]
Culture of the transformant is performed by a method known per se. Culture
conditions, for example, temperature, pH of the medium, and culture time are
suitably
selected. For example, when the host is an animal cell, an MEM medium
(Science,
Vol.122, p.501, 1952), DMEM medium (Virology, Vol.8, p.396, 1959), RPMI1640
medium
(J. Am. Med. Assoc., Vol.199, p.519, 1967), 199 medium (Proc. Soc. Exp. Biol.
Med.,
Vol.73, p.1, 1950) and the like containing about 5% to 20% fetal bovine serum
can be used
as the medium. The pH of the medium is preferably about 6 to 8, culture is
normally
performed at about 30 C to 40 C for about 15 to 72 hours, and aeration or
agitation may be
performed as necessary. When the host is an insect cell, for example, Grace's
medium
(Proc. Natl. Acad. Sci. USA, Vol.82, p.8404, 1985) and the like containing
fetal bovine
serum can be mentioned, and the pH thereof is preferably about 5 to 8.
Culturing is
normally performed at about 20 C to 40 C for 15 to 100 hours, and aeration or
agitation
may be performed as necessary. When the host is a bacterium, an actinomyces,
yeast, or
a filamentous fungus, for example, a liquid medium containing the above-
described
nutrient sources is appropriate. A medium having a pH of 5 to 8 is preferable.
When the
host is E. coli, preferred examples of the medium include LB medium, M9 medium
(Miller
et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory, p.431, 1972) and the
like. In this
case, culture can be normally performed at 14 C to 43 C for about 3 to 24
hours, while
aeration or agitation is performed as necessary. When the host is a bacterium
of the genus
14
CA 02859627 2014-06-17
Bacillus, cultivation can be normally performed at 30 C to 40 C for about 16
to 96 hours,
while aeration or agitation is performed as necessary. When the host is yeast,
examples
of the medium include Burkholder's minimal medium (Bostian, Proc. Natl. Acad.
Sci.
USA, Vol.77, p.4505, 1980), and the pH of the medium is desirably 5 to 8.
Culturing is
normally performed at about 20 C to 35 C for about 14 to 144 hours, and
aeration or
agitation may be performed as necessary.
[0049]
By culturing a transformant as described above, the anti-human CTGF antibody
of
the present invention can be recovered, preferably isolated and purified, from
the
transformant. Examples of the method of isolation and purification include
methods
based on differences in solubility, such as salting-out and solvent
precipitation; methods
based on differences in molecular weight, such as dialysis, ultrafiltration,
gel filtration, and
sodium dodecyl sulfate-polyacrylamide gel electrophoresis; methods based on
electric
charge, such as ion exchange chromatography and hydroxyl apatite
chromatography;
methods based on specific affinity, such as affinity chromatography; methods
based on
differences in hydrophobicity, such as reverse phase high performance liquid
chromatography; methods based on differences in isoelectric point, such as
isoelectric
focusing; and the like.
[0050]
Although the present invention has been generally described above, specific
examples are provided herein only for a better understanding of the present
invention.
These examples are for illustrative purposes only and do not limit the scope
of the present
invention.
[Examples]
[0051]
The procedures involving the use of a kit or a reagent and the like were
performed
in accordance with the attached protocol attached unless otherwise stated.
[0052]
(Example 1: Acquisition of CTGF Protein Derived from Various Sources)
The present inventors acquired a human CTGF protein as an antigen for
preparing
an anti-CTGF antibody. The full-length gene (SEQ ID NO: 13) of the human CTGF
was
ligated into an expression vector (pcDNA3.1; Invitrogen), and the vector thus
prepared was
genetically introduced into a FreeStyle 293 cell (Invitrogen) using a
FreeStyle MAX
Reagent (Invitrogen) as a gene introducing reagent. This cell was cultured in
a serum-
free culture system using a FreeStyle 293 Expression Medium (Invitrogen), and
then a
culture supernatant including the human CTGF protein was acquired. Protein was
purified from the culture supernatant thus acquired, using a HiTrap heparin
column and a
CM column (GE Healthcare Japan), and then used in the experiment as follows.
Mouse,
CA 02859627 2014-06-17
rat and monkey CTGF proteins were acquired using the same method.
[0053]
(Example 2: Immunization of VelocImmune Mouse)
An antibody for human CTGF was acquired by immunization for a VelocImmune
mouse. In order to increase the diversity of an antibody to be obtained, the
present
inventors have investigated a plurality of immunization methods,
administration routes,
adjuvants, immune periods, and the like. As an immunogen, purified human CTGF
was
used and mixed with an adjuvant to perform immunization. As the administration
route,
footpad administration and intraperitoneal administration were investigated.
As the
adjuvant, TiterMax Gold (CytRx Corporation), a complete Freund's adjuvant
(Sigma), and
an incomplete Freund's adjuvant (Sigma) were investigated. In
addition, as an
immunestimulant to be added, CpG oligonucleotide and Aluminum Phosphate Gel
(manufactured by BRENNTAG) were investigated. As for the immunization period,
the
immunization was performed for 3 weeks to 14 weeks. After performing
immunization
several times, mice were subjected to blood sampling from caudal vein to
monitor a titer,
and thus, VelocImmune mice that produce antibodies binding to human CTGF were
chosen.
[0054]
The titration was measured using a standard ELISA method below. 20 1.1L of
phosphate buffer physiological saline (PBS) solution of human CTGF (1 g/mL)
was
added to a Maxisorp 384 plate (Nunc, Inc.), and immobilized by being incubated
overnight
at 4 C. The next day, the plate was washed once with 100 jtL of washing
solution
(TBST: 0.05% Tween-20-containing Tris buffer), and then 100 jtL of blocking
agent (1%
BSA-containing PBS) was added thereto and allowed to stand at room temperature
for 1
hour. After washing once with 100 pi, of TBST washing solution, a series of
dilutions of
plasma in the sampled blood were prepared and added thereto. After incubation
at room
temperature for 1 hour, and washing with 100 L of TBST washing solution three
times, a
goat anti-mouse IgG antibody labeled with a horseradish peroxidase (HRP-goat
anti-mouse
IgG antibody; Zymed Laboratories, Inc.) that had been diluted 5000-fold with a
0.1%
BSA-containing TBST washing solution (20 1AL) was added thereto. After
incubation at
room temperature for 1 hour, washing with 100 JAL of TBST washing solution was
conducted three times. After adding 40 1AL of TMB chromogenic reagent
(Sumitomo
Bakelite Co., Ltd.) thereto and allowing it to stand at room temperature for
10 minutes, 40
iAL of stopping solution (2 mol/L sulfuric acid) was added thereto to stop the
reaction, and
the absorbance at 450 nm was measured.
[0055]
(Example 3: Preparation of Anti-human CTGF Antibody-Producing Hybridoma)
Final immunization (intravenous administration or intraperitoneal
administration
of an antigen) was carried out for a mouse chosen by checking the increase in
the antibody
16
CA 02859627 2014-06-17
titer. A hybridoma was prepared by collecting lymphocytes by removing spleen
and
lymph nodes of immunized mice according to a conventional method, and cell-
fusing them
into a mouse myeloma cell SP2/0. The hybridoma was subjected to limiting
dilution and
monocloning, and then the antibody was purified from the supernatant using a
protein A or
protein G column (GE Healthcare Japan).
[0056]
(Example 4: ELISA Assay)
The present inventors evaluated the binding specificity of the antibody for
CTGF
using an ELISA method. 20 pL of PBS solution of human CTGF (1 lig/mL) was
added
to a Maxisorp 384 plate (Nunc, Inc.), and immoblized by being incubated
overnight at 4 C.
The next day, the plate was washed once with 100 'IL of washing solution
(TPBS: 0.05%
Tween-20-containing PBS), and then 100 piL of blocking agent (1% BSA-
containing PBS)
was added thereto and allowed to stand at room temperature for 1 hour. After
washing
once with 100 laL of TBST washing solution, a series of appropriate dilutions
of the
purified antibody sample were prepared and added to the plate. After
incubation at room
temperature for 1 hour, the plate was washed three times with 100 lit, of TBST
washing
solution, and a goat anti-mouse IgG antibody labeled with a horseradish
peroxidase (HRP-
goat anti-mouse IgG antibody; Zymed Laboratories, Inc.) which was diluted 5000-
fold
with a 0.1% BSA-containing TBST washing solution (20 !AL) was added thereto.
After
incubation at room temperature for 1 hour, the plate was washed three times
with 100 1.1L
of TBST washing solution. After adding 401AL of TMB chromogenic reagent
(Sumitomo
Bakelite Co., Ltd.) thereto and allowing it to stand at room temperature for
10 minutes, 40
piL of stopping solution (2 mol/L sulfuric acid) was added thereto to stop the
reaction, and
the absorbance at 450 nm was measured. Each of the antibodies was tested in
duplicate,
and the EC50 was analyzed by curve fitting.
[0057]
As a result, it was confirmed that an antibody referred to as 37-45 has high
binding activity (EC50: 1.6 ng/ml).
[0058]
(Example 5: Sequencing of Antibody)
For the identified 37-45 antibody, the present inventors cloned a gene
encoding
the heavy chain and light chain of the antibody from the hybridoma. RNA was
extracted
from the hybridoma, and a cDNA was prepared using a cDNA amplification kit
(SMARTer
RACE cDNA Amplification kit; Clontech). Subsequently, the variable regions of
the
heavy chain and light chain were elongated and amplified using PCR. The PCR
products
were recombined with a vector for subcloning PCR products such as pCR3.1-TOPO
(Invitrogen), and then the gene was sequenced using a sequencer (ABI PRISM
3100;
Applied Biosystems).
17
CA 02859627 2014-06-17
[0059]
The determined base sequence of the heavy-chain variable region of 37-45 is
shown by SEQ ID NO: 1 and the amino acid sequence thereof is shown by SEQ ID
NO: 2,
and the determined base sequence of the light-chain variable region of 37-45
is shown by
SEQ ID NO: 3 and the amino acid sequence thereof is shown by SEQ ID NO: 4.
[0060]
(Example 6: Preparation of Fully Human Antibody)
For the above-described antibody, the variable region is derived from a human
and the constant region is derived from a mouse. Therefore, the present
inventors
replaced the constant region derived from a mouse by the constant region
derived from a
human to prepare a fully human antibody (fully human 37-45). Specifically, a
signal
sequence was linked to the 5' side of the heavy-chain variable region gene of
the antibody
and the constant region gene of human Igyl (Man Sung Co., etc. (1992) J
Immunol. Vol.
148 (4): 1149-1154) was linked to the 3' side of the heavy-chain variable
region gene of the
antibody. The heavy-chain gene was inserted into a GS vector (Lonza Biologics)
pEE6.4.
Upon insertion, a restriction enzyme BbvCI recognizing site in the gene was
converted to a
DNA sequence that does not affect the amino acid sequence of the antibody. In
addition,
a signal sequence was linked to the 5' side of the light-chain variable region
gene of the
antibody and the constant region gene of human i chain (Man Sung Co., etc.,
supra) was
linked to the 3' side of the light-chain variable region gene of the antibody.
The light-
chain gene was inserted into a GS vector pEE12.4.
[0061]
For the heavy chain of the prepared fully human 37-45, the base sequence is
shown by SEQ ID NO: 5 and the amino acid sequence is shown by SEQ ID NO: 6,
and for
the light chain of the antibody, the base sequence is shown by as SEQ ID NO: 7
and the
amino acid sequence is shown by SEQ ID NO: 8.
[0062]
(Example 7: Preparation of Variant of Glycosylation Site of Variable Region)
The amino acid of the heavy-chain variable region of fully human 37-45 as
described above includes an N-type glycosylation motif sequence of N-X-(T/S).
Specifically, in the heavy-chain variable region shown by SEQ ID NO: 2, Asn at
the
position 58 according to Kabat numbering corresponds to the glycosylation
site. If the
glycosylation site is present, addition of sugar chains to the antibody occurs
during cell
culture, but it is known that the addition of sugar chains depends on culture
conditions or
hosts for expression. That is, even with the same antibody-producing cells
thus
established, there is a possibility that a degree of the addition of sugar
chains varies
according to culture conditions (a medium, a cell concentration, and the
like), and there is
also a possibility that it is difficult to acquire an antibody medical product
having uniform
18
CA 02859627 2014-06-17
quality. Therefore, the present inventors prepared a fully human antibody
(fully human
37-45-MH1) in which mutations had been introduced to the heavy-chain variable
region of
fully human 37-45.
[0063]
For the heavy-chain variable region of the prepared fully human 37-45-MH1, the
base sequence is shown by SEQ ID NO: 9 and the amino acid sequence is shown by
SEQ
ID NO: 10. For the heavy chain of the prepared fully human 37-45-MH1, the base
sequence is shown by SEQ ID NO: 11 and the amino acid sequence is shown by SEQ
ID
NO: 12. The light chain of fully human 37-45-MH1 is the same as the light
chain of fully
human 37-45.
[0064]
The CDR1, CDR2 and CDR3 of the heavy-chain variable region of fully human
37-45-MH1 antibody is a region of position from 31 to 35, 50 to 65, and 95 to
102 of the
heavy-chain variable region based on Kabat numbering, respectively, which
consists of the
amino acid sequence at position from 31 to 35, 50 to 66, and 99 to 108 of SEQ
ID NO:10,
respectively. The CDR1, CDR2 and CDR3 of the light-chain variable region of
fully
human 37-45-MH1 antibody is a region of position from 24 to 34, 50 to 56, and
89 to 97 of
the light-chain variable region based on Kabat numbering, respectively, which
consists of
the amino acid sequence at position from 24 to 35, 51 to 57, and 90 to 98 of
SEQ ID NO:4,
respectively.
[0065]
(Example 8: Expression and Purification of Fully Human Antibody)
The GS vector in which the genes of the heavy chain and light chain of each
antibody as described above, fully human 37-45 and fully human 37-45-MH1, has
been
inserted was cleaved with restriction enzymes, NotI and PvuI, and ligated
using a Ligation-
Convenience Kit (NIPPONGENE) or a Ligation-high (TOYOBO) to construct a GS
vector
in which both genes of the heavy chain and light chain had been inserted. This
vector
encodes the full-length heavy and light chains, and a glutamine synthetase,
and it was
transfected into CHO-K1SV cells to express an antibody. The culture
supernatant was
purified with a Protein A or Protein G column (GE Healthcare Japan) to obtain
a purified
antibody of each fully human antibody.
[0066]
(Example 9: ELISA Assay of Fully Human Antibody)
The present inventors evaluated the binding specificity of fully human 37-45
and
fully human 37-45-MH1 prepared in the above Examples to human, mouse, rat and
monkey CTGF using an ELISA method. Here, the same method as described in
Example
4 was used, but a rabbit anti-human IgG antibody labeled with horseradish
peroxidase
(HRP-rabbit anti-human IgG antibody; DAKO) which was 5000-fold diluted with a
0.1%
19
CA 02859627 2014-06-17
BSA-containing TBST washing solution as a secondary antibody. The test on each
antibody was carried out in duplicate and EC50 was analyzed by curve fitting.
[0067]
As a result, it was found that all of fully human antibodies had the same
degrees
of binding ability for human, mouse, rat and monkey CTGF.
Table 1: Binding Activity of Fully Human Antibody for Various CTGF
[Table 1]
Fully human 37-45 Fully
human 37-45-MH1
EC50 (ng/ml) EC50 (ng/ml)
Human CTGF 13.2 10.4
Mouse CTGF 12.4 9.2
Rat CTGF 13.1 9.2
Monkey CTGF 12.5 8.6
[0068]
(Example 10: Evaluation of Binding Activity by SPR Analysis)
In order to measure the antigen-specific binding activity of fully human 37-45-
MH1 in more detail, the present inventors carried out surface plasmon
resonance (SPR)
analysis. In the present Example, an anti-human CTGF antibody CLN1 (Patent
Document 2) was used as a comparative antibody.
[0069]
In the SPR analysis, Biacore 2000 (GE Healthcare Japan) was used to carry out
analysis. An anti-CTGF antibody was immobilized on the surface of a Sensor
Chip CM5
using a Human Antibody Capture Kit and an Amine Coupling Kit (GE Healthcare
Japan).
Serial dilution of the human CTGF acquired in Example 1 was made by HBS-EP
solution
(GE Healthcare Japan). 100 pt,1_, of the dilution was added to flow path at
flow rate 50
By this measurement system, the association rate constant (ka), the
dissociation
rate constant (kd), and the dissociation constant (KD) between the human CTGF
protein
and the anti-CTGF antibody were calculated using a data analysis software (BIA
Evaluation).
Table 2: Binding Activity to human CTGF of Fully Human 37-45-MH1 by SPR
Analysis
[Table 2]
KD (M) Kd (1/s)
Fully human 37-45-MH1 3.7x10-11 1.6x104
CLN1 4.6x10-1 3.7x10-3
CA 02859627 2014-06-17
[0070]
As a result, it was found that fully human 37-45-MH1 has about 12 times or
higher binding activity for human CTGF than that of the antibody CLN1.
[0071]
(Example 11: Inhibitory Action on Collagen Synthesis in Rat Kidney-derived
Cells)
The present inventors investigated the inhibitory effect on TGF13-induced
collagen
synthesis in the rat fibroblast NRK-49F in order to measure the antigen-
specific
neutralizing action of fully human 37-45-MH1. In the present Example, CLN1 was
used
as a comparative antibody.
[0072]
NRK-49F cells (available from ATCC) produce CTGF by the addition of TGF13.
The NRK-49F cells were seeded into a 24-well plate in a 10% FCS-containing
DMEM
medium (5x104 cells), and after 24 hours, the medium was replaced with a 0.01%
FCS-
containing DMEM (500 4). Further, after 24 hours, TGFP (R&D Systems; 1 ng/ml)
was added to the medium. At 1 hour before the addition of TGFB, anti-human
CTGF
antibodies, fully human 37-45-MH1 or CLN1, were added (to three groups at 1
fig/ml, 3
pig/m1 and 10 ptg/m1). After 72 hours, the supernatant was recovered and
subjected to
SDS-PAGE, and Western Blot analysis was carried out according to an ordinary
method
using an Anti-Collagen I antibody (Abcam plc). As a result, it was found that
fully
human 37-45-MH1 has a strong ability of inhibiting collagen synthesis, as
compared with
CLN1 in a concentration-dependent manner.
[0073]
(Example 12: Evaluation Test on Kidney Function by Mouse Remnant Kidney
Model)
Glomerulosclerosis and renal tubular degeneration are a finding, which appears
commonly in a variety of renal disorders causing chronic renal diseases. These
chronic
renal diseases can be investigated in the mouse remnant kidney model
exhibiting
progressive renal disorders. In this model, a load is applied to the residual
kidney by 2/3
unilateral nephrectomy and contralateral total nephrectomy (5/6 nephrectomy),
thereby
inducing proteinuria and significant reduction in the functions of the kidney,
and
histopathological glomerular sclerosis or renal tubular degeneration is shown
and mild
interstitial fibrosis is shown (see, for example, Kidney International, 64,
350-355, 2003).
[0074]
5/6 Nephrectomy was carried out with reference to a method of Zhang, et al.
(Kidney International, 56, 549-558, 1999). A 9-week-old male mouse ICR (Japan
SLC,
Inc., Hamamatsu-shi, Shizuoka-ken) was anesthetized by the intraperitoneal
administration
21
CA 02859627 2014-06-17
of pentobarbital (50 mg/kg), and the head 1/3 and the caudal 1/3 of the left
kidney were
resected. One week after the first surgery, the mouse was anesthetized by
intraperitoneal
administration of pentobarbital (50 mg/kg), and the right kidney was
completely removed
to complete the 5/6 nephrectomy.
[0075]
Urine collection and blood sampling were performed one week after the 5/6
nephrectomy, and urinary protein excretion rate and renal function parameters
(serum
creatinine concentration and creatinine clearance) were measured. The
protein
concentration measurement was performed by a Bradford method (Bio-Rad
Laboratories).
The creatinine concentration was measured using CRE-EN Kainos (Kainos
Laboratories,
Inc.). The urinary protein excretion rate was calculated by correcting the
urinary protein
concentration (mg/ml) with the urinary creatinine concentration (mg/dL). The
urinary
protein excretion rate, the serum creatinine concentration, and the creatinine
clearance
were taken as indicators, and thus, the groups were divided into solvent-
treated group
(administration of a phosphate buffer with pH 7.4) and antibody administration
group (15
mice per group). The tests started by setting the doses of antibodies to three
groups, 0.5
mg/kg, 1 mg/kg and 2 mg/kg. The phosphate buffer and fully human 37-45-MH1
were
injected subcutaneously into the back once a week (six doses in total). At the
start of the
test, at weeks 4 and 6 from the start of the test, the urine samples and the
blood samples
were collected, and the urinary protein excretion rate, the serum creatinine
concentration,
and the creatinine clearance were measured.
[0076]
For the urinary protein excretion rate, at a time of the start of the test, in
the
solvent-treated group, the urinary protein excretion rate increased, as
compared with the
normal group (normal group 5.1 0.4; solvent-treated group 9.7 0.7 (P<0.01)).
Also, at
weeks 4 and 6 from the start of the test, in the solvent-treated group, the
urinary protein
excretion rate increased, as compared with the normal group. On the other
hand, in the
antibody-treated groups (1 mg/kg group and 2 mg/kg group), although there was
no
statistically significant difference, the urinary protein excretion rate
decreased in a dose-
dependent manner, as compared with the solvent-treated group.
[0077]
For the serum creatinine concentration, at a time of the start of the test, in
the
solvent-treated group, the serum creatinine concentration increased, as
compared with the
normal group (normal group 0.36 0.013 mg/dL; solvent-treated group 0.53 0.016
mg/dL
(P<0.01)). Thereafter, also at weeks 4 and 6, in the solvent-treated group,
the serum
creatinine concentration increased, as compared with the normal group (week 4:
normal
group 0.42 0.025 mg/dL; solvent-treated group 0.66 0.037 mg/dL (P<0.01), week
6:
normal group 0.31 0.016 mg/dL; solvent-treated group 0.81 0.126 mg/dL
(P<0.05)). In
22
CA 02859627 2014-06-17
the antibody-treated groups, for the 0.5 mg/kg group, although there was no
significant
difference, the serum creatinine concentration decreased at weeks 4 and 6, as
compared
with the solvent-treated group. In addition, in the 1 mg/kg group and the 2
mg/kg group,
the increase in the serum creatinine concentration was significantly
inhibited, as compared
with the solvent-treated group (week 4: 1 mg/kg group 0.51 0.022 mg/dL
(P<0.05); 2
mg/kg group 0.51 0.015 mg/dL (P<0.05), week 6: 1 mg/kg group 0.55 0.043 mg/dL
(P<0.05); 2 mg/kg group 0.49 0.024 mg/dL (P<0.01)).
[0078]
For the creatinine clearance (urinary creatinine concentrationxamount of urine
for
24 hours/serum creatinine concentration), at a time of the start of the test,
in the solvent-
treated group, the decrease in the creatinine clearance was confirmed as
compared with the
normal group (normal group 1.8 0.18; solvent-treated group 1.3 0.08 (P<0.01)).
Thereafter, also at weeks 4 and 6, in the solvent-treated group, the
creatinine clearance
decreased as compared with the normal group (week 4: normal group 2.1 0.16;
solvent-
treated group 1.6 0.16, week 6: normal group 2.8 0.29; solvent-treated group
1.4 0.17
(P<0.001)). For the antibody-treated groups, in the 0.5 mg/kg group,
inhibition of the
decrease in the creatinine clearance was not confirmed as compared with the
solvent-
treated group. On the other hand, in the 1 mg/kg group, at weeks 4 and 6, the
decrease in
the creatinine clearance was significantly inhibited, as compared with the
solvent-treated
group (week 4: solvent-treated group 1.6 0.16; 1 mg/kg group 2.1 0.11
(P<0.05), week 6:
solvent-treated group 1.4 0.17; 1 mg/kg group 2.0 0.18 (13<0.05)). In
addition, in the 2
mg/kg group, at week 6, the decrease in the creatinine clearance was
significantly inhibited,
as compared with the solvent-treated group (week 6: solvent-treated group 1.4
0.17; 2
mg/kg group 1.9 0.14 (P<0.05)).
[0079]
From these results, it was confirmed that fully human 37-45-MH1 inhibits
reduction in the renal functions in a chronic kidney disease model.
[0080]
(Example 13: Pharmacological Evaluation Test on Rat Nephritis Models)
Rat anti-Thy 1.1 models are established mesangial proliferative
glomerulonephritis models, with the pathological conditions expressed by the
injection of
antibodies to Thy antigens on the surface of mesangial cells in the renal
glomeruli (see, for
example, Yamamoto and Wilson, 1987 Kidney Int. 32:514-25, Morita, et al., 1998
Am J
Kidney Dis 31:559-73). In the present models, after the lysis of the mesangial
cells,
mesangial cell proliferation and extracellular matrices increase, and the
level of urine
protein is enhanced (see, for example, Floege, et al., 1991 Kidney Int. 40:477-
88, Ito, et al.,
2001 J Am Soc Nephrol. 12:472-84). The anti-Thy 1.1 models are similar to IgA
nephropathy or Henoch¨Schonlein purpura in human, and the progress of the
pathological
23
CA 02859627 2014-06-17
conditions can be predicted using the models with proteinuria as an indicator
(see, for
example, Kasuga, et al., 2001 Kidney Int. 60:1745-55, Liu, et al., 2007
Nephron Exp
Nephrol. 105:e65-74).
[0081]
A solution of anti-Thy 1.1 antibody (Anti-Rat CD90 (Thy 1.1) monoclonal
antibody-ascites; CEDARLANE) was prepared by physiological saline at 0.1 g/mL.
Nephritis was expressed by intravenously administering the antibody solution
to rats (200
per 100 g body weight). After 4 hours from the administration of anti-Thy 1.1
antibodies, fully human 37-45-MH1 (0.5 mg/kg, 1 mg/kg or 2 mg/kg) or solvent
(PBS)
were intravenously administered. Urine collection were performed for 24 hours
after 3 to 4
days from the inducement of the pathogenesis, and the urinary protein
excretion amount in
24 hours (UP) and the urinary protein excretion rate (UP/uCr: the urinary
protein
concentration (mg/ml) was corrected with the urinary creatinine concentration
(mg/dL))
were measured. The results are shown in Table 3 (UP) and Table 4 (UP/uCr).
24
CA 02859627 2014-06-17
Table 3: UP
[Table 3]
Inhibitory rate (%) vs
UP (mg/day) p value
solvent-administered group
Normal animal group 1.9 100.0
Solvent-administered
114.3 0.0 p<0.001
#
group (PBS)
Fully human 37-45-MH I
115.2 -0.8
0.5 mg/kg
Fully human 37-45-MH1
95.5 16.8
1 mg/kg
Fully human 37-45-MH1
83.8 27.2 p=0.029 *
2 mg/kg
#: vs normal animal group by t-test
*: vs solvent-administered group by Dunnett's test
Table 4: UP/uCr
[Table 4]
UP/uCr Inhibitory rate (%) vs
p value
(mg/mg) solvent-administered group
Normal animal group 0.315 100.0
Solvent-administered
33.865 0.0 p<0.001 #
group (PBS)
Fully human 37-45-MH1
26.280 22.6
0.5 mg/kg
Fully human 37-45-MH1
22.487 33.9 p=0.037 *
1 mg/kg
Fully human 37-45-MH1
18.427 46.0 p=0.0039 *
2 mg/kg
#: vs normal animal group by t-test
*: vs solvent-administered group by Dunnett's test
25
CA 02859627 2014-06-17
[0082]
As a result, fully human 37-45-MH1 inhibited the proteinuria in a dose-
dependent
manner, and the inhibitory rates at 2 mg/kg group were 27.2% and 46.0% vs
solvent-
administered group, respectively, with the indices of UP and UP/uCr.
[0083]
Next, for the purpose of identifying the difference from CLN1 in the action
strength, the same models were used for evaluation. The evaluation procedure
was the
same as above. For the evaluation, with reference to the doses which were
effective in
the above, 2 mg/kg of fully human 37-45-MH1 was used as a positive control,
and 2 mg/kg
26
CA 02859627 2014-06-17
Table 5: UP
[Table 5]
Inhibitory rate Inhibitory rate
UP (%) vs solvent- (%) vs IgG-
p value p value
(mg/day) administered administered
group group
Normal animal
0.8 100.0 100.0
group
Solvent-
administered 111.1 0.0 p<0.001 #
group (PBS)
Control IgG
114.5 -3.1 0.0 p<0.001 #
2 mg/kg
Control IgG
113.4 -2.1 0.0 p<0.001 #
20 mg/kg
CLN1
97.2 12.6 p=0.45 * 15.2 p=0.22 &
2 mg/kg
CLN1
107.3 3.5 p=0.79 * 5.4 p=0.53 $
20 mg/kg
Fully human
37-45-MH1 79.0 29.1 p=0.044 * 31.2 p=0.0011 &
2 mg/kg
#: vs normal animal group by t-test
*: vs solvent-administered group by t-test
&: vs Control IgG 2 mg/kg group by t-test
$: vs Control IgG 20 mg/kg group by t-test
27
CA 02859627 2014-06-17
Table 6: UP/uCr
[Table 6]
Inhibitory rate Inhibitory rate
UP/uCr (%) vs solvent- (%) vs IgG-
p value p
value
(mg/mg) administered administered
group group
Normal animal
0.3 100.0 100.0
group
Solvent-
administered 46.1 0.0 p<0.001 #
group (PBS)
Control IgG
52.2 -13.4 0.0 p<0.001 #
2 mg/kg
Control IgG
42.7 7.5 0.0 p<0.001 #
20 mg/kg
CLN1
36.3 21.5 p=0.18 * 30.8 p=0.030 &
2 mg/kg
CLN1
41.8 9.5 p=0.49 * 2.2 p=0.82 $
20 mg/kg
Fully human
37-45-MH1 28.1 39.2 p=0.0092 * 46.4
p<0.001 &
2 mg/kg
#: vs normal animal group by t-test
*: vs solvent-administered group by t-test
[0084]
As a result, the pathological conditions were expressed in substantially the
same
degree as the previous experiment. In addition, 2 mg/kg of fully human 37-45-
MH1
showed substantially the same inhibitory rate as evaluated in the previous
experiment, and
the inhibitory rates were 29.1% and 39.2%, respectively, with the indices of
UP and
UP/uCr, in the case of using the solvent-administered group as a control.
[0085]
On the other hand, CLN1 had a less inhibitory action on proteinuria, as
compared
28
CA 02859627 2014-06-17
with the fully human 37-45-MH1 (the inhibitory rates vs solvent-administered
group were
12.6% and 21.5%, respectively, with the indices of UP and UP/uCr at 2 mg/kg,
and the
inhibitory rates vs solvent-administered group were 3.5% and 9.5%,
respectively, with the
indices of UP and UP/uCr at 20mg/kg). Further, for the human IgG1 antibodies,
there
was a substantially little action on proteinuria.
[0086]
From this, it was confirmed that fully human 37-45-MH1 has a strong
proteinuria
inhibitory action, as compared with CLN1.
[Industrial Applicability]
[0087]
The anti-human CTGF antibody of the present invention is useful for prevention
or treatment of various diseases that human CTGF is involved in pathogenesis,
in a range
of renal diseases such as chronic kidney disease and diabetic nephropathy.
29
